We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33 C and 37 C) and reduced at room temperature (22 C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions characterized by single-stranded RNA flanked by structured elements and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and therefore presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2.

Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.

Sexual development in the human fungal pathogen Cryptococcus neoformans is a multistep process that results in the formation of spores, the likely infectious particles. A critical step in this developmental process is the transition from bud-form growth to filamentous growth. This transition is controlled by the homeodomain transcription factors Sxi1␣ and Sxi2a, whose targets are largely unknown. Here we describe the discovery of a gene, CLP1, that is regulated by Sxi1␣ and Sxi2a and is essential for sexual development. In vitro binding studies also show that the CLP1 promoter is bound directly by Sxi1␣ and Sxi2a. The deletion of CLP1 leads to a block in sexual development after cell fusion but before filament formation, and cells without CLP1 are unable to grow vegetatively after cell fusion. Our findings lead to a model in which CLP1 is a downstream target of the Sxi proteins that functions to promote growth after mating and to establish the filamentous state, a critical step in the production of spores.

BackgroundSerum interleukin 6 (IL‐6), chemokine ligand 2 (CCL2), C‐reactive protein (CRP), and the ratio of aspartate transaminase to alanine transaminase (AST:ALT) have been correlated with fibrosis and necroinflammatory activity in humans with various hepatopathies.Hypothesis/ObjectivesTo determine whether increases in serum IL‐6, CCL2, CRP, or AST:ALT were associated with moderate to severe fibrosis or necroinflammatory activity in dogs with various hepatopathies.AnimalsForty‐four client‐owned dogs with clinical evidence of liver disease and 10 healthy purpose‐bred dogs, all undergoing liver biopsies by laparoscopy or laparotomy.MethodsMeasurement of serum IL‐6, CCL2, CRP, AST, and ALT before scheduled liver biopsy and evaluation of liver histopathology using the METAVIR scoring system used in human medicine, blinded to clinical presentation.ResultsMedian serum IL‐6 was approximately twice as high in dogs with high fibrosis scores (15.5 pg/mL; range, 1.4 to 235 pg/mL) compared to dogs with low fibrosis scores (7.6 pg/mL; range, 1.4 to 148.1 pg/mL), with marginal significance (P = .05). Median serum CCL2 was significantly higher in dogs with active necroinflammation (444 pg/mL; range, 144 to 896 pg/mL) compared to dogs without detectable necroinflammation (326 pg/mL; range, 59 to 1692 pg/mL; P = .008), but with considerable overlap between groups. Neither serum CRP nor AST:ALT ratios were significantly different based on fibrosis or necroinflammatory scores.Conclusions and Clinical ImportanceBecause of substantial variability among dogs, single measurements of IL‐6 and CCL2 have limited diagnostic utility for identifying fibrosis or necroinflammation, respectively, in dogs with various chronic liver diseases. The value of these biomarkers should be explored further in monitoring response to treatment in individual dogs with chronic hepatopathies.

Introduction Transitional cell carcinoma (TCC) in humans is associated with environmental exposures and variants in glutathione S‐transferase (GST) genes. Scottish Terriers have a high breed risk for TCC, but the relationship between genetic and environmental risk in dogs is not fully understood. Hypotheses Scottish Terriers have a higher frequency of GST‐theta variants compared to lower risk breeds. Dogs with TCC of any breed have a higher frequency of GST‐theta variants along with higher environmental exposures, compared to controls. Animals One hundred and five Scottish Terriers and 68 controls from lower risk breeds; 69 dogs of various breeds with TCC, and 72 breed‐ and sex‐matched unaffected geriatric dogs. Methods In this prospective case‐control study, dogs were genotyped for 3 canine GST‐theta variants: GSTT1 I2+28 G>A, a GSTT1 3′UTR haplotype, and GSTT5 Asp129_Gln130del. Owners of dogs with TCC and unaffected geriatric controls completed a household environmental questionnaire. Results The GSTT1 3′UTR haplotype and GSTT5 Asp129_Gln130del variants were significantly underrepresented in Scottish Terriers (minor allele frequency [MAF] = 0.000 for both), compared to dogs from lower risk breeds (MAF = 0.108 and 0.100; P ≤ .0002). Dogs with TCC did not differ from unaffected geriatric controls across the 3 investigated loci. Transitional cell carcinoma was associated with household insecticide use (odds ratio [OR] = 4.28, 95% confidence interval [CI] = 1.44‐12.33, P = .02), and was negatively associated with proximity to a farm (OR = 0.49, 95% CI = 0.25‐0.99, P = .04). Conclusions and Clinical Importance Low‐activity GST‐theta loci are unlikely contributors to TCC risk in dogs. Increased risk is associated with household insecticide use, and possibly with less rural households.