In enteric viral infections, such as those with rotavirus and norovirus, individual viral particles shed in stool are considered the optimal units of fecal-oral transmission. We reveal that rotaviruses and noroviruses are also shed in stool as viral clusters enclosed within vesicles that deliver a high inoculum to the receiving host. Cultured cells non-lytically release rotaviruses and noroviruses inside extracellular vesicles. In addition, stools of infected hosts contain norovirus and rotavirus within vesicles of exosomal or plasma membrane origin. These vesicles remain intact during fecal-oral transmission and thereby transport multiple viral particles collectively to the next host, enhancing both the MOI and disease severity. Vesicle-cloaked viruses are non-negligible populations in stool and have a disproportionately larger contribution to infectivity than free viruses. Our findings indicate that vesicle-cloaked viruses are highly virulent units of fecal-oral transmission and highlight a need for antivirals targeting vesicles and virus clustering.

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone including its tissue remodeling and antifibrotic effects. The peptide hormone has a short half-life in plasma, limiting its therapeutic utility. However, small molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach especially for chronic diseases such as heart failure and fibrosis. The first small molecule agonists of RXFP1 were recently identified from a high throughput screen using a homogenous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seven transmembrane domain (TM7) and the third extracellular loop (ECL3). A number of residues were found to play an important role in the agonist binding and receptor activation including a hydrophobic region at TM7 consisting of W664, F668 and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule agonist ML290 were compared with the cognate ligand relaxin peptide, providing valuable insights on the structural basis and molecular mechanism of activation and selectivity of the agonists for RXFP1.

Relaxin, a small peptide hormone of the insulin/relaxin family, demonstrated antifibrotic, organ protective, vasodilatory, and proangiogenic properties in clinical trials and several animal models of human diseases. Relaxin family peptide receptor 1 (RXFP1) is the relaxin cognate G protein-coupled receptor. We have identified a series of small molecule agonists of human RXFP1. The lead compound ML290 demonstrated preferred absorption, distribution, metabolism, and excretion profiles, is easy to synthesize, and has high stability in vivo. However, ML290 does not activate rodent RXFP1s and therefore cannot be tested in common preclinical animal models. Here we describe the production and analysis of a mouse transgenic model, a knock-out/knock-in of the human RXFP1 (hRXFP1) complementary DNA into the mouse Rxfp1 (mRxfp1) gene. Insertion of the vector into the mRxfp1 locus caused disruption of mRxfp1 and expression of hRXFP1. The transcriptional expression pattern of the hRXFP1 allele was similar to mRxfp1. Female mice homozygous for hRXFP1 showed relaxation of the pubic symphysis at parturition and normal development of mammary nipples and vaginal epithelium, indicating full complementation of mRxfp1 gene ablation. Intravenous injection of relaxin led to an increase in heart rate in humanized and wild-type females but not in Rxfp1-deficient mice, whereas ML290 increased heart rate in humanized but not wild-type animals, suggesting specific target engagement by ML290. Moreover, intraperitoneal injection of ML290 caused a decrease in blood osmolality. Taken together, our data show humanized RXFP1 mice can be used for testing relaxin receptor modulators in various preclinical studies.

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit–human and guinea pig–human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.