Electroacupuncture (EA) stimulates somatic median afferents underlying P5-6 acupoints and modulates parasympathoexcitatory reflex responses through central processing in the brainstem. Although decreases in blood pressure and heart rate by the neural-mediated Bezold-Jarisch reflex responses are modulated by EA through opioid actions in the nucleus tractus solitarius and nucleus ambiguus, the role of the hypothalamus is unclear. The hypothalamic paraventricular nucleus (PVN) is activated by sympathetic afferents and regulates sympathetic outflow and sympathoexcitatory cardiovascular responses. In addition, the PVN is activated by vagal afferents, but little is known about its regulation of cardiopulmonary inhibitory hemodynamic responses. We hypothesized that the PVN participates in the Bezold-Jarisch reflex responses and EA inhibits these cardiopulmonary responses through the PVN opioid system. Rats were anesthetized and ventilated, and their heart rate and blood pressures were monitored. Application of phenylbiguanide every 10 min close to the right atrium induced consistent depressor and bradycardia reflex responses. Unilateral microinjection of the depolarization blockade agent kainic acid or glutamate receptor antagonist kynurenic acid in the PVN reduced these reflex responses. In at least 70% of the rats, 30 min of bilateral EA at P5-6 acupoints reduced the depressor and bradycardia responses for at least 60 min. Blockade of the CCK-1 receptors converted the non-responders into EA-responders. Unilateral PVN-microinjection with naloxone reversed the EA inhibition. Vagal-evoked activity of the PVN cardiovascular neurons was reduced by 30 min EA (P5-6) through opioid receptor activation. These data indicate that PVN processes inhibitory cardiopulmonary reflexes and participates in EA-modulation of the neural-mediated vasodepression and bradycardia.

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-)protein into ribonucleoprotein particles (RNPs), 38 ± 10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to ancestral and mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining nucleocapsid protein variants in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multivalent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N-protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.