Cell-free assays reveal the HIV-1 capsid protects reverse transcripts from cGAS.

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect their genomes from cGAS even after completion of reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, capsid inhibitors also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

Next-generation mapping of the salicylic acid signaling hub and transcriptional cascade.

For over 60 years, salicylic acid (SA) has been known as a plant immune signal required for both basal and systemic acquired resistance (SAR). SA activates these immune responses by reprogramming up to 20% of the transcriptome through the function of NPR1. However, components in the NPR1-signaling hub, which appears as nuclear condensates, and the NPR1- signaling cascade remained elusive due to difficulties in studying transcriptional cofactors whose chromatin associations are often indirect and transient. To overcome this challenge, we applied TurboID to divulge the NPR1-proxiome, which detected almost all known NPR1-interactors as well as new components of transcription-related complexes. Testing of new components showed that chromatin remodeling and histone demethylation contribute to SA-induced resistance. Globally, NPR1-proxiome shares a striking similarity to GBPL3-proxiome involved in SA synthesis, except associated transcription factors (TFs), suggesting that common regulatory modules are recruited to reprogram specific transcriptomes by transcriptional cofactors, like NPR1, through binding to unique TFs. Stepwise greenCUT&RUN analyses showed that, upon SA-induction, NPR1 initiates the transcriptional cascade primarily through association with TGA TFs to induce expression of secondary TFs, predominantly WRKYs. WRKY54 and WRKY70 then play a major role in inducing immune-output genes without interacting with NPR1 at the chromatin. Moreover, a loss of NPR1 condensate formation decreases its chromatin-association and transcriptional activity, indicating the importance of condensates in organizing the NPR1- signaling hub and initiating the transcriptional cascade. This study demonstrates how combinatorial applications of TurboID and stepwise greenCUT&RUN transcend traditional genetic methods to globally map signaling hubs and transcriptional cascades.

Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics.

California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA.IMPORTANCEOrthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

Structural basis of NPR1 in activating plant immunity.

NPR1 is a master regulator of the defence transcriptome induced by the plant immune signal salicylic acid1-4. Despite the important role of NPR1 in plant immunity5-7, understanding of its regulatory mechanisms has been hindered by a lack of structural information. Here we report cryo-electron microscopy and crystal structures of Arabidopsis NPR1 and its complex with the transcription factor TGA3. Cryo-electron microscopy analysis reveals that NPR1 is a bird-shaped homodimer comprising a central Broad-complex, Tramtrack and Bric-à-brac (BTB) domain, a BTB and carboxyterminal Kelch helix bundle, four ankyrin repeats and a disordered salicylic-acid-binding domain. Crystal structure analysis reveals a unique zinc-finger motif in BTB for interacting with ankyrin repeats and mediating NPR1 oligomerization. We found that, after stimulation, salicylic-acid-induced folding and docking of the salicylic-acid-binding domain onto ankyrin repeats is required for the transcriptional cofactor activity of NPR1, providing a structural explanation for a direct role of salicylic acid in regulating NPR1-dependent gene expression. Moreover, our structure of the TGA32-NPR12-TGA32 complex, DNA-binding assay and genetic data show that dimeric NPR1 activates transcription by bridging two fatty-acid-bound TGA3 dimers to form an enhanceosome. The stepwise assembly of the NPR1-TGA complex suggests possible hetero-oligomeric complex formation with other transcription factors, revealing how NPR1 reprograms the defence transcriptome.

Development of HEK-293 Cell Lines Constitutively Expressing Flaviviral Antigens for Use in Diagnostics.

Flaviviruses are important human pathogens worldwide. Diagnostic testing for these viruses is difficult because many of the pathogens require specialized biocontainment. To address this issue, we generated 39 virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells of 13 different flaviviruses, including dengue, yellow fever, Japanese encephalitis, West Nile, St. Louis encephalitis, Zika, Rocio, Ilheus, Usutu, and Powassan viruses. Antigen secretion was stable for at least 10 cell passages, as measured by enzyme-linked immunosorbent assays and immunofluorescence assays. Thirty-five cell lines (90%) had stable antigen expression over 10 passages, with three of these cell lines (7%) increasing in antigen expression and one cell line (3%) decreasing in antigen expression. Antigen secretion in the HEK-293 cell lines was higher than in previously developed COS-1 cell line counterparts. These antigens can replace current antigens derived from live or inactivated virus for safer use in diagnostic testing. IMPORTANCE Serological diagnostic testing for flaviviral infections is hindered by the need for specialized biocontainment for preparation of reagents and assay implementation. The use of previously developed COS-1 cell lines secreting noninfectious recombinant viral antigen is limited due to diminished antigen secretion over time. Here, we describe the generation of 39 flaviviral virus-like particle (VLP)- and nonstructural protein 1 (NS1)-secreting stable cell lines in HEK-293 cells representing 13 medically important flaviviruses. Antigen production was more stable and statistically higher in these newly developed cell lines than in their COS-1 cell line counterparts. The use of these cell lines for production of flaviviral antigens will expand serological diagnostic testing of flaviviruses worldwide.

Monoclonal antibodies to Cache Valley virus for serological diagnosis.

Cache Valley virus (CVV) is a mosquito-borne virus in the genus Orthobunyavirus, family Peribunyaviridae. It was first isolated from a Culiseta inorata mosquito in Cache Valley, Utah in 1956 and is known to circulate widely in the Americas. While only a handful of human cases have been reported since its discovery, it is the causative agent of fetal death and severe malformations in livestock. CVV has recently emerged as a potential viral pathogen causing severe disease in humans. Currently, the only serological assay available for diagnostic testing is plaque reduction neutralization test which takes several days to perform and requires biocontainment. To expand diagnostic capacity to detect CVV infections by immunoassays, 12 hybridoma clones secreting anti-CVV murine monoclonal antibodies (MAbs) were developed. All MAbs developed were found to be non-neutralizing and specific to the nucleoprotein of CVV. Cross-reactivity experiments with related orthobunyaviruses revealed several of the MAbs reacted with Tensaw, Fort Sherman, Tlacotalpan, Maguari, Playas, and Potosi viruses. Our data shows that MAbs CVV14, CVV15, CVV17, and CVV18 have high specific reactivity as a detector in an IgM antibody capture test with human sera.

Quantitative Proteomics Reveals a Role for SERINE/ARGININE-Rich 45 in Regulating RNA Metabolism and Modulating Transcriptional Suppression via the ASAP Complex in Arabidopsis thaliana.

Pre-mRNA alternative splicing is a conserved mechanism for eukaryotic cells to leverage existing genetic resources to create a diverse pool of protein products. It is regulated in coordination with other events in RNA metabolism such as transcription, polyadenylation, RNA transport, and nonsense-mediated decay via protein networks. SERINE/ARGININE-RICH 45 (SR45) is thought to be a neutral splicing regulator. It is orthologous to a component of the apoptosis and splicing-associated protein (ASAP) complex functioning to regulate RNA metabolism at multiple levels. Within this context, we try to understand why the sr45-1 mutant Arabidopsis has malformed flowers, delayed flowering time, and increased disease resistance. Prior studies revealed increased expression for some disease resistance genes and the flowering suppressor Flowering Locus C (FLC) in sr45-1 mutants and a physical association between SR45 and reproductive process-related RNAs. Here, we used Tandem Mass Tag-based quantitative mass spectrometry to compare the protein abundance from inflorescence between Arabidopsis wild-type (Col-0) and sr45-1 mutant plants. A total of 7,206 proteins were quantified, of which 227 proteins exhibited significantly different accumulation. Only a small percentage of these proteins overlapped with the dataset of RNAs with altered expression. The proteomics results revealed that the sr45-1 mutant had increased amounts of enzymes for glucosinolate biosynthesis which are important for disease resistance. Furthermore, the mutant inflorescence had a drastically reduced amount of the Sin3-associated protein 18 (SAP18), a second ASAP complex component, despite no significant reduction in SAP18 RNA. The third ASAP component protein, ACINUS, also had lower abundance without significant RNA changes in the sr45-1 mutant. To test the effect of SR45 on SAP18, a SAP18-GFP fusion protein was overproduced in transgenic Arabidopsis Col-0 and sr45-1 plants. SAP18-GFP has less accumulation in the nucleus, the site of activity for the ASAP complex, without SR45. Furthermore, transgenic sr45-1 mutants overproducing SAP18-GFP expressed even more FLC and had a more severe flowering delay than non-transgenic sr45-1 mutants. These results suggest that SR45 is required to maintain the wild-type level of SAP18 protein accumulation in the nucleus and that FLC-regulated flowering time is regulated by the correct expression and localization of the ASAP complex.

Immunopathologic Effects of Prednisolone and Cyclosporine A on Feline Immunodeficiency Virus Replication and Persistence.

Feline immunodeficiency virus (FIV) induces opportunistic disease in chronically infected cats, and both prednisolone and cyclosporine A (CsA) are clinically used to treat complications such as lymphoma and stomatitis. However, the impact of these compounds on FIV infection are still unknown and understanding immunomodulatory effects on FIV replication and persistence is critical to guide safe and effective therapies. To determine the immunologic and virologic effects of prednisolone and CsA during FIV infection, FIV-positive cats were administered immunosuppressive doses of prednisolone (2 mg/kg) or CsA (5 mg/kg). Both prednisolone and CsA induced acute and transient increases in FIV DNA and RNA loads as detected by quantitative PCR. Changes in the proportion of lymphocyte immunophenotypes were also observed between FIV-infected and naïve cats treated with CsA and prednisolone, and both treatments caused acute increases in CD4+ lymphocytes that correlated with increased FIV RNA. CsA and prednisolone also produced alterations in cytokine expression that favored a shift toward a Th2 response. Pre-treatment with CsA slightly enhanced the efficacy of antiretroviral therapy but did not enhance clearance of FIV. Results highlight the potential for drug-induced perturbation of FIV infection and underscore the need for more information regarding immunopathologic consequences of therapeutic agents on concurrent viral infections.

Feline Leukemia Virus (FeLV) Disease Outcomes in a Domestic Cat Breeding Colony: Relationship to Endogenous FeLV and Other Chronic Viral Infections.

Exogenous feline leukemia virus (FeLV) is a feline gammaretrovirus that results in a variety of disease outcomes. Endogenous FeLV (enFeLV) is a replication-defective provirus found in species belonging to the Felis genus, which includes the domestic cat (Felis catus). There have been few studies examining interaction between enFeLV genotype and FeLV progression. We examined point-in-time enFeLV and FeLV viral loads, as well as occurrence of FeLV/enFeLV recombinants (FeLV-B), to determine factors relating to clinical disease in a closed breeding colony of cats during a natural infection of FeLV. Coinfections with feline foamy virus (FFV), feline gammaherpesvirus 1 (FcaGHV-1), and feline coronavirus (FCoV) were also documented and analyzed for impact on cat health and FeLV disease. Correlation analysis and structural equation modeling techniques were used to measure interactions among disease parameters. Progressive FeLV disease and FeLV-B presence were associated with higher FeLV proviral and plasma viral loads. Female cats were more likely to have progressive disease and FeLV-B. Conversely, enFeLV copy number was higher in male cats and negatively associated with progressive FeLV disease. Males were more likely to have abortive FeLV disease. FFV proviral load was found to correlate positively with higher FeLV proviral and plasma viral load, detection of FeLV-B, and FCoV status. Male cats were much more likely to be infected with FcaGHV-1 than female cats. This analysis provides insights into the interplay between endogenous and exogenous FeLV during naturally occurring disease and reveals striking variation in the infection patterns among four chronic viral infections of domestic cats.IMPORTANCE Endogenous retroviruses are harbored by many animals, and their interactions with exogenous retroviral infections have not been widely studied. Feline leukemia virus (FeLV) is a relevant model system to examine this question, as endogenous and exogenous forms of the virus exist. In this analysis of a large domestic cat breeding colony naturally infected with FeLV, we documented that enFeLV copy number was higher in males and inversely related to FeLV viral load and associated with better FeLV disease outcomes. Females had lower enFeLV copy numbers and were more likely to have progressive FeLV disease and FeLV-B subtypes. FFV viral load was correlated with FeLV progression. FFV, FcaGHV-1, and FeLV displayed markedly different patterns of infection with respect to host demographics. This investigation revealed complex coinfection outcomes and viral ecology of chronic infections in a closed population.

Transcriptome analyses reveal SR45 to be a neutral splicing regulator and a suppressor of innate immunity in Arabidopsis thaliana.

BACKGROUND: Regulation of pre-mRNA splicing diversifies protein products and affects many biological processes. Arabidopsis thaliana Serine/Arginine-rich 45 (SR45), regulates pre-mRNA splicing by interacting with other regulatory proteins and spliceosomal subunits. Although SR45 has orthologs in diverse eukaryotes, including human RNPS1, the sr45-1 null mutant is viable. Narrow flower petals and reduced seed formation suggest that SR45 regulates genes involved in diverse processes, including reproduction. To understand how SR45 is involved in the regulation of reproductive processes, we studied mRNA from the wild-type and sr45-1 inflorescences using RNA-seq, and identified SR45-bound RNAs by immunoprecipitation. RESULTS: Using a variety of bioinformatics tools, we identified a total of 358 SR45 differentially regulated (SDR) genes, 542 SR45-dependent alternative splicing (SAS) events, and 1812 SR45-associated RNAs (SARs). There is little overlap between SDR genes and SAS genes, and neither set of genes is enriched for flower or seed development. However, transcripts from reproductive process genes are significantly overrepresented in SARs. In exploring the fate of SARs, we found that a total of 81 SARs are subject to alternative splicing, while 14 of them are known Nonsense-Mediated Decay (NMD) targets. Motifs related to GGNGG are enriched both in SARs and near different types of SAS events, suggesting that SR45 recognizes this motif directly. Genes involved in plant defense are significantly over-represented among genes whose expression is suppressed by SR45, and sr45-1 plants do indeed show enhanced immunity. CONCLUSION: We find that SR45 is a suppressor of innate immunity. We find that a single motif (GGNGG) is highly enriched in both RNAs bound by SR45 and in sequences near SR45- dependent alternative splicing events in inflorescence tissue. We find that the alternative splicing events regulated by SR45 are enriched for this motif whether the effect of SR45 is activation or repression of the particular event. Thus, our data suggests that SR45 acts to control splice site choice in a way that defies simple categorization as an activator or repressor of splicing.