Molecular phylogeny of coronaviruses and host receptors among domestic and close-contact animals reveals subgenome-level conservation, crossover, and divergence.

BACKGROUND: Coronaviruses have the potential to cross species barriers. To learn the molecular intersections among the most common coronaviruses of domestic and close-contact animals, we analyzed representative coronavirus genera infecting mouse, rat, rabbit, dog, cat, cattle, white-tailed deer, swine, ferret, mink, alpaca, Rhinolophus bat, dolphin, whale, chicken, duck and turkey hosts; reference or complete genome sequences were available for most of these coronavirus genera. Protein sequence alignments and phylogenetic trees were built for the spike (S), envelope (E), membrane (M) and nucleocapsid (N) proteins. The host receptors and enzymes aminopeptidase N (APN), angiotensin converting enzyme 2 (ACE2), sialic acid synthase (SAS), transmembrane serine protease 2 (TMPRSS2), dipeptidyl peptidase 4 (DPP4), cathepsin L (and its analogs) and furin were also compared. RESULTS: Overall, the S, E, M, and N proteins segregated according to their viral genera (α, ß, or γ), but the S proteins of alphacoronaviruses lacked conservation of phylogeny. Interestingly, the unique polybasic furin cleavage motif found in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) but not in severe acute respiratory syndrome coronavirus (SARS-CoV) or Middle East respiratory syndrome coronavirus (MERS-CoV) exists in several ß-coronaviruses and a few α- or γ-coronaviruses. Receptors and enzymes retained host species-dependent relationships with one another. Among the hosts, critical ACE2 residues essential for SARS-CoV-2 spike protein binding were most conserved in white-tailed deer and cattle. CONCLUSION: The polybasic furin cleavage motif found in several ß- and other coronaviruses of animals points to the existence of an intermediate host for SARS-CoV-2, and it also offers a counternarrative to the theory of a laboratory-engineered virus. Generally, the S proteins of coronaviruses show crossovers of phylogenies indicative of recombination events. Additionally, the consistency in the segregation of viral proteins of the MERS-like coronavirus (NC_034440.1) from pipistrelle bat supports its classification as a ß-coronavirus. Finally, similarities in host enzymes and receptors did not always explain natural cross-infections. More studies are therefore needed to identify factors that determine the cross-species infectivity of coronaviruses.

Interference with pathways activated by topoisomerase inhibition alters the surface expression of PD-L1 and MHC I in colon cancer cells.

Topoisomerase inhibitors are clinically used to treat various cancer types, including colorectal cancer. These drugs also activate signaling pathways that modulate cell survival and immune cell functions. Immunotherapy is promising for certain tumors, including microsatellite instable colorectal cancer, but not for microsatellite stable colorectal cancer. The reasons for this lack of responsiveness are largely unknown. Understanding how colorectal cancer cell-surface proteins interact with tumor-resident immune cells may offer an opportunity to identify molecules that, if targeted, may render tumor cells visible to immune cells. The present study used flow cytometry, fluorescent staining and immunoblotting to examine if inhibition of pathways activated by topoisomerase-targeting drugs may modulate the outcomes of treatment through effects on cell cycle arrest and apoptosis, and by altering surface expression levels of programmed death-ligand 1 (PD-L1) or major histocompatibility complex protein I (MHC I). Inhibition of either NF-κB or DNA-damage response (DDR) potently enhanced cell death in combination with topoisomerase inhibition, while only NF-κB inhibition increased MHC I. PD-L1 upregulation was moderately affected by NF-κB or DDR inhibitors, while both topoisomerase inhibitors and DNA damaging agents may enhance the surface expression of MHC I molecules on colon cancer cells. Such enhanced expression of MHC I may be suppressed by inhibitors of ataxia-telangiectasia mutated or checkpoint kinase kinases. Additionally, adaptive tolerance to topoisomerase inhibition caused altered cell cycle response, and reduced the expression levels of both PD-L1 and MHC I on both microsatellite instable and stable colon cancer cell lines. Therefore, targeted modulation of DDR pathways, PD-L1, MHC I or other immune regulators in colon cancer cells may make them more visible to immune cells and enable rational combination of conventional therapy with immunotherapy.