Structure of anellovirus-like particles reveal a mechanism for immune evasion.

Anelloviruses are nonpathogenic viruses that comprise a major portion of the human virome. Despite being ubiquitous in the human population, anelloviruses (ANVs) remain poorly understood. Basic features of the virus, such as the identity of its capsid protein and the structure of the viral particle, have been unclear until now. Here, we use cryogenic electron microscopy to describe the first structure of an ANV-like particle. The particle, formed by 60 jelly roll domain-containing ANV capsid proteins, forms an icosahedral particle core from which spike domains extend to form a salient part of the particle surface. The spike domains come together around the 5-fold symmetry axis to form crown-like features. The base of the spike domain, the P1 subdomain, shares some sequence conservation between ANV strains while a hypervariable region, forming the P2 subdomain, is at the spike domain apex. We propose that this structure renders the particle less susceptible to antibody neutralization by hiding vulnerable conserved domains while exposing highly diverse epitopes as immunological decoys, thereby contributing to the immune evasion properties of anelloviruses. These results shed light on the structure of anelloviruses and provide a framework to understand their interactions with the immune system.

Discovery of GS-7682, a Novel 4'-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

Mechanism and spectrum of inhibition of a 4'-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRps) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus and human metapneumovirus incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.