Highlights d SARS-CoV-2 spike evolves during persistent infection to resist common antibodies d Antibody affinity maturation is critical to neutralization breadth d Intra-host evolution foreshadows mutations in circulating spike variants
Alphaviruses, like many other arthropod-borne viruses, infect vertebrate species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described 1 – 3 , most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2–E1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBD–Fc fusion protein or a ligand-binding antagonist block Semliki forest virus E2–E1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR LBD–Fc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis.
The RNA-dependent RNA polymerase (RdRP) of nonsegmented negative-sense RNA viruses consists of a large catalytic protein (L) and a phosphoprotein cofactor (P). During infection, the RdRP replicates and transcribes the viral genome, which resides inside an oligomer of nucleocapsid protein (N-RNA). The classical view of P as a cofactor for L assigns a primary role of P as a bridge mediating the access of L to the RNA template, whereby its N-terminal domain (P NTD ) binds L and its C-terminal domain (P CTD ) binds N-RNA. Recent biochemical and structural studies of a prototype nonsegmented negative-sense RNA virus, vesicular stomatitis virus, suggest a role for P beyond that of a mere physical link: P induces a structural rearrangement in L and stimulates polymerase processivity. In this study, we investigated the critical requirements within P mediating the functional interaction with L to form a fully functional RdRP. We analyzed the correlation between the impact of P on the conformation of L and its activity in RNA synthesis and the consequences of these events on RdRP function. We identified three separable elements of the P NTD that are required for inducing the conformational rearrangement of L, stimulating polymerase processivity, and mediating transcription of the N-RNA. The functional interplay between these elements provides insight into the role of P as a dynamic player in the RNA synthesis machine, influencing essential aspects of polymerase structure and function.large polymerase | replication and transcription | Mononegavirales | rhabdovirus T he functional unit necessary for transcription and replication of nonsegmented negative-sense (NNS) RNA viruses is a ribonucleoprotein (RNP) complex. The RNP complex comprises a genomic RNA encapsidated by a nucleocapsid protein oligomer (N-RNA), associated with the RNA-dependent RNA polymerase (RdRP) consisting of a complex of the large polymerase protein (L) and a phosphoprotein (P) (1-3). The template RNA is buried between the N-and C-terminal lobes of each N protomer; nevertheless, the overall integrity of the N-RNA structure is maintained during copying by the RdRP (4, 5). The L protein is the multifunctional catalytic core of the RNA synthesis machinery, harboring the RdRP as well as a capping enzyme and two methyltransferase activities that are required for mRNA synthesis (6-10). During RNA synthesis, L must gain access to the RNA, and its enzymatic activities must be regulated in accordance with a replicase or transcriptase mode of RNA synthesis. The access of L to the N-RNA is mediated by the noncatalytic cofactor P, which engages L and the N oligomer simultaneously (11,12).The functioning of an RNP as a highly regulated RNA synthesis machine requires an intricate, tight coordination of its individual components. The mechanisms that govern such functional coupling are largely unknown. Much of our understanding of the assembly, structure, and function of NNS RNA virus RNPs have come from studies of vesicular stomatitis virus (VSV). In part, this reflect...
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